Introduction
As the most prevalent gastrointestinal cancer, colorectal cancer has a continuously increasing incidence in young people with poor prognosis [
1]. Many pathological and molecular therapies have been explored to serve as clinical interventions for colorectal cancer, which achieved much progress [
2,
3]. However, the improvements in survival of colorectal cancer patients are still unsatisfactory [
4].
Tumor cells often show intense abilities of proliferation, migration, invasion, anti-apoptosis, angiogenesis, and avoidance of growth inhibition. Recently, it has been found that tumors also can alter energy metabolism, which contributes a lot to helping the cells maintain the malignancy and making the glycolytic signaling pathway another essential feature of cancer [
5]. Cancerous cells commonly display glycolytic pathway activation, which keeps the cells in high metabolic status and produces more lactic acid, resulting in decreasing extracellular pH [
6]. High levels of glycolysis and low extracellular pH are associated with malignancy, including cancer cell proliferation and invasion, leading to a worse prognosis in colorectal cancer patients [
7,
8].
Long non-coding RNA (lncRNA), with over 200 nucleotides in length, participates in many physiological and pathological processes, including immune response [
9], growth and development [
10], the incidence of diverse diseases, for instance, cardiovascular disease, neurodegenerative disease, and cancer [
11]. LncRNA could modulate gene expression by working as microRNA sponges [
12]. Lots of lncRNA play their roles in cancer genesis and development, such as HOTAIRs [
13], MEG3 [
14], MALAT-1 [
15], H19 [
16], and GCLNC1 [
17]. Recently, thymopoietin-antisense RNA 1 (TMPO-AS1), functioning as a novel regulator to play roles in cancer, has drawn much attention [
18,
19]. However, its roles in colorectal cancer haven't been clarified yet.
Here, we found that TMPO-AS1 regulated glycolysis in colorectal cancer by modulating miR-1270/PKM2 axis. This research illustrated the oncogenic effect of TMPO-AS1 and raised a new insight into the molecular mechanism of colorectal cancer.
Materials and Methods
Specimens
Totally 30 colorectal tumor tissues and the corresponding 30 adjacent normal tissues involved in the project collected from October 2022 to February 2023. were from the patients signing with informed consent at the First Affiliated Hospital of Harbin Medical University. All these patients haven’t received any chemotherapy or preoperative radiotherapy before. The tissue samples were kept in liquid nitrogen immediately after the surgical removal for subsequent detection. All experiments were permitted by the Ethics Committee.
Cell culture and transfection
The colorectal cancer cell lines SW480 (Shanghai, Zhong Qiao Xin Zhou Biotechnology) were cultured in complete DMEM supplementary with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37℃-incubator supplement with 5% CO2. All the culture reagents, including the culture medium, penicillin/streptomycin, FBS, and trypsin, were purchased from Hyclone Laboratories, Inc. (UT, USA). For cell transfection, the lncRNA TMPO-AS1 siRNAs, and the relative control siRNAs were synthesized by Genscript Biotech (Nanjing, China). MiR-1270 mimics, miR-1270 inhibitors, and the relative control mimics and inhibitors were provided by Ribo Bio (Guangzhou, China). The sequence of si-TMPO-AS1 is 5’-GCU UGU CUG CAG GCA CUC AUA-3'; the si-control is 5’-GCG UUG CUG UCG CUC UAU UCU-3'. All siRNAs and miRNA mimics or inhibitors were performed transfection to cells for 48 h using Lipofectamine 3000 (Invitrogen, NY, USA).
Cell viability assay
Applying the Cell Counting Kit-8 assay kit (CCK-8, Yeasen Bio, Shanghai, China), the SW480 cell proliferative rate was evaluated by detecting the change in cell viability. Seeded the cells in a 96-well plate first, and supplied CCK-8 solution 10 μL to each well simultaneously every day. After another 2 h of incubation, the absorbance with a microplate reader (Molecular Devices, CA, USA) at 450 nm.
Flow cytometry
After transfection of siRNAs or miRNA mimics or inhibitors, SW480 cells were obtained. Rinse the cells twice by PBS, and dye them with FITC-AnnexinV and PI. Placed the mixtures for 15 min at room temperature in the dark, and then evaluated the cell apoptosis profile on the BD FACS Calibur (BD Bioscience, CA, USA).
Wound-healing assay
After the transfection of siRNAs or miRNA mimics or inhibitors was indicated, the cells were made a scratch using the tips, and pictures of the scratch on the plate were taken. 48 h later, took pictures of the scratch on the plate again. Recorded the width of the scratch at 0 h and 48 h, and calculated the wound closure.
Transwell assay
The cell invasion ability was evaluated via transwell assay. We carried out this experiment using the chambers containing 8.0-mm pore membranes (Millipore, Billerica, USA). We firstly used 200 μL of fetal bovine serum-free DMEM to re-suspend the SW480 cells and then plated all the cells as mentioned above into the top chamber pre-coated with a matrigel (Solarbio Life Sciences, Beijing, China). After that, we supplied a 0.5 mL DMEM containing 20% FBS to the bottom chamber as a chemoattractant for cells. 48 h later, the invaded cells were fixed for 30 min and stained with crystal violet solution for about 20 min. Next, we used the inverted microscope to take a photograph and count the quantity of the stained cells.
Reverse transcription-quantitative PCR
Extracted total RNAs of SW480 cells by Trizol reagent (Invitrogen, NY, USA). After the extracted RNAs were quantified and purified, cDNA was obtained by transcribing 5 μg RNA. Following, qRT-PCR was implemented with SYBR qPCR Taq (Takara, Tokyo, Japan) under the procedure of 95 ℃ for 6 min, and 40 circles of the process of 95℃ for 60 s, 58℃ for 30 s, 72℃ for 60 s, followed with 72℃ for 10 min. Gene level was tested via the 2 −ΔΔCT method. The internal control refers to U6 and GAPDH. The primer sequences are as follows: TMPO-AS1-F: 5’-AGC CCA CAC ACT ACA GGC A-3'; TMPO-AS1-R: 5'- GCA CAA AAG CAG TAC GAC CT -3'; miR-1270-F: 5’-GGA GAT ATG GAA GAG CTG TGT-3'; miR-1270-R: 5’-CGA TCA GCA TTT CCA ATA TGC A-3'; PKM2-F: 5'- ACT GGC ATC ATC TGT ACC ATT G -3'; PKM2-R: 5'- AGC CAC ATT CAT TCC AGA CTT A -3'; U6-F: 5’-CGC AGC GGC AGC GGA TAT AC-3'; GAPDH-R: 5’-AAT GAA GGG GTC ATT GAT GG-3'; U6-R: 5’-ACG AAT TTG CGT GTC ATC CTT GCG-3'; GAPDH-F: 5’-GAG TCA ACG GAT TTG GTC GT-3'.
Western blot
Extracted the proteins from SW480 cells with the radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Merk Life Sciences, Darmstadt, Germany) supplementary with phosphatase inhibitor and protease inhibitor (Sigma-Aldrich, Shanghai, China). The concentration of the extracted proteins was assessed using a commercialized Bradford protein assay kit (BOSTER Biotechnology, Wuhan, China). Protein of the same quantity (50 μg) was electrophoresly separated on 12% SDS-PAGE gel, and the protein in gels was transferred to methanol-pretreated PVDF membranes (Millipore, Darmstadt, Germany). The membranes with separated proteins on them were then submerged with 5% skim milk solution at 25℃ for 90 min, and immersed in indicated diluted primary antibodies at 4 °C for at least 12 h, followed by incubating with indicated secondary antibodies at the dilution of 1:5000 at room temperature for 90 min. In the present study, the primary antibodies against PCNA, Ki67, MMP2, and MMP9 were offered by Abcam (Cambridge, UK). Antibody against β-actin (Proteintech, Wuhan, China) was the internal control. Diluted all primary antibodies at the ratio of 1:1000. The protein bands were observed by the chemiluminescence in the enhanced ECL immunoblotting system (Tanon, Shanghai, China) and analyzed by ImageJ.
Glucose uptake assay
Glucose Uptake Colorimetric Assay Kit (Biovision, CA, USA) was applied for the measurement of cell glucose uptake accordingly. The SW480 cells were inoculated in a 96-well dish (1500 cells per well). Before the detection, the cells were removed from the medium rinsed twice by 1 × PBS, and cultured in an FBS-free medium overnight. The other day, after the cells 3 times with 1 × PBS were washed, the cells were subjected to starvation from glucose for 40 min by the (Krebs–Ringer-Phosphate-HEPES) KRPH buffer. Then 10 μL 2- deoxyglucose (2-DG) was mixed into the reaction system for 20 min, followed by 1 h of enzyme mixture incubation. Added the extraction buffer and the Neutralizing buffer and then added the mixture of glutathione reductase and DTNB. Read the absorbance at 412 nm by using an automatic microplate reader (Molecular Devices, CA, USA).
Lactate production assay
The Lactate Colorimetric Assay Kit (Elabscience, Wuhan, China) was utilized to measure the lactate produced by cells under the instruction of the manufacturer. Briefly, the cell culture supernatant (50 μL) was added to the microplate plate, then a 50 μL lactate enzyme mixture was mixed with the samples and kept at 25℃ for 25 min. Finally, read the absorbance at 450 nm by using the automatic microplate reader.
ATP production assay
The ATP produced by cells was examined utilizing the commercialized ATP detection kit (Elabscience, Wuhan, China). Briefly, the cells using lysis buffer in the kit, performed 10 min of centrifugation at 11,000 g, and removed the supernatant. 50 μL samples were added to a 96-well dish, followed by an addition with a working solution at a volume of 100 μL. Next, the luminescence of the mixture was detected by an automatic microplate reader (BioTek, Winooski, VT, USA).
Dual-luciferase reporter assay
Starbase was utilized for the prediction of the TMPO-AS1 binding sites of miR-1270 and the miR-1270 binding sites of PKM2. The pMIR-GLO report luciferase vector was obtained from Genepharma (Shanghai, China). The wild type (WT) of the seed sequence of miR-1270 in TMPO-AS1 or 3'UTR of PKM2 and the mutant type (Mut) of the seed sequence were magnified and implanted into the pMIR-GLO vector to construct wild type and mutant type luciferase plasmid. The wild type or the mutant type plasmids were co-transfected with the control mimic or miR-1270 mimic to SW480 cells for 48 h via Lipofectamine 3000 (Invitrogen, NY, USA). The luciferase signals were detected with a commercialized assay kit (Yeasen Biotechnology, Shanghai, China) and normalized to the Ranilla luciferase activity.
Starbase database analysis
We applied Starbase v3.0 (
http://starbase.sysu.edu.cn/) database to obtain the potential binding miRNA of TMPO-AS1. In this study, the binding of miRNA of TMPO-AS1 was predicted by Starbase v3.0.
Statistical analysis
GraphPad Prism (7.0 version) was applied to perform the statistics in this project. Means ± Standard Deviation presented all data, and all trials were conducted 3 times. One-way ANOVA or Unpaired Student t-test was for testing the significant differences between pair groups, and Fisher's LSD test was for multiple groups. P < 0.05 means statistically significant.
Discussion
In recent years, lncRNAs have been found to participate in the metastasis, progression and programmed cell death of diverse cancers, including colorectal cancer [
25,
26]. lncRNAs function as a clinical predictive marker of colorectal cancer through biological regulation of cell metastasis, proliferation, and apoptosis [
27]. Besides, glucose metabolism’s role in colorectal cancer development has attracted researchers [
28]. However, the studies of lncRNA in regulating glucose metabolism in colorectal tumors are still limited. This research clarified that TMPO-AS1 regulated glycolysis in colorectal cancer and exerted carcinogenesis via mediating miR-1270/PKM2 axis.
TMPO-AS1 draws much attention to its function in cancers, including breast cancer, bladder cancer, and pancreatic cancer. TMPO-AS1 was reported to upregulate the tumor tissues in colorectal cancer and correlated closely to metastasis [
29]. Consistently, TMPO-AS1 was enriched in the colorectal tumor tissues, which predicted a poor prognosis for colorectal cancer patients in this research. Furthermore, TMPO-AS1 knockdown restrained proliferation, decreased the migrative and invasive abilities, and accelerated apoptosis in SW480 cells, following the previous reports of TMPO-AS1's oncogenic effect on other types of cancer cells [
30,
31].
Glucose metabolism plays a vital role in tumor progression. Inhibition of glucose metabolism contributes to tumor cell death [
23,
24], although some experimental evidence indicates that increased glucose metabolism may be related to stronger immune therapy resistance [
32,
33]. Tumor cells require more aerobic glycolysis to produce ATP, with more lactate production and glucose uptake, called the Warburg effect [
34]. In this study, the knockdown of TMPO-AS1 decreased ATP production, cell lactate secretion, and glucose uptake, indicating that TMPO-AS1 negatively regulated the glycolysis of the colorectal cancer cells. LncRNAs often played their roles through sponging to miRNAs to work as the competitive endogenous RNA of miRNAs, contributing to the inhibition of miRNA function. We confirmed that TMPO-AS1 was directly bound to miR-1270 by luciferase reporter assay and qRT-PCR. MiR-1270 is a tumor-suppressive miRNA [
35,
36]. Accordingly, in this project, overexpression of miR-1270 suppressed the proliferative and invasive abilities and the glucose metabolism of tumor cells. Besides, we noticed that miR-1270 directly targeted to 3'UTR of PKM2 to downregulate the expression of PKM2. PKM2, coding M1 and M2 type of pyruvate kinase (PK), which participates in glycolysis, is the primary subtype of PK expressed in cancer cells [
37]. PKM2 showed an oncogenic effect to promote cancer progression [
38]. In this study, miR-1270 inhibitors abolished si-TMPO-AS1’s effect on suppressing the tumor cell proliferation, invasion, and glycolysis, while knockdown of PKM2 further reversed miR-1270 inhibitors effect on the TMPO-AS1 knockdown, indicating that TMPO-AS1 regulated tumor cell malignancy and glucose metabolism through miR-1270/PKM2 axis. We did not study the immune therapy resistance affected by the inhibition of glucose metabolism so further investigation is demanded to make clear the mechanism of tumor cell malignancy and glucose metabolism.
Moreover, Studies by Khanmi Kasomva et al. have shown that miRNAs, as primary regulators of mrna, can regulate the metabolic reprogramming of cancer cells either directly or indirectly by interacting with the glycolysis process during tumorigenesis [
39,
40]. It has been found that mirnas can not only regulate glucose transporters (GLUTs/SLC2A), which is a key protein that helps glucose enter cells and control glycolysis, but also miRNA targeting GLUT is believed to play a role in regulating GLUT expression and enhancing glycolysis in a variety of cancer types such as prostate cancer [
41] and liver cancer [
42,
43].
In addition, mirnas can regulate a large number of enzymes involved in glucose, fatty acids, and amino acids metabolism, which are easily reprogrammed in cancer cells to meet higher metabolic demands. For example, the key regulator of the Warburg effect [
44,
45], hexokinase (HK)2, a glycolytic enzyme found to be overexpressed in tumors, has been shown to enhance glycolysis and alter glucose metabolism in various cancers such as liver cancer cells [
46] and breast cancer [
47] by miRNA regulation [
48]. In addition, glycolytic enzymes such as phosphofructokinase 1(PFK1) [
49] and hepatic phosphofructokinase (PFKL) [
50] are also regulated by miRNAs in cancer.
In a word, the present study provides a basis to clarify the relationship between TMPO-AS1 and the malignant degree of tumor cells and glucose metabolism.. Mechanically, TMPO-AS1 could increase PKM2 expression by playing as a competing endogenous RNA through binding to miR-1270 directly. This study provides a theoretical basis for TMPO-AS1/miR-1270/PKM2 regulatory network to become a new biomarker for the treatment of colorectal cancer.
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